Cynara cardunculus as a reducing and capping agent in this study demonstrated the possibility of green manufacturing of silver nanoparticles (CC-AgNPs). UV–visible spectroscopy validated the development of CC-AgNPs, indicating the surface plasmon resonance (SPR) λmax band at 438 nm. The band gap of CC-AgNPs was found to be 2.26 eV. SEM and TEM analysis examined the surface morphology of CC-AgNPs, and micrographs revealed that the nanoparticles were spherical. The crystallinity, crystallite size, and phase purity of as-prepared nanoparticles were confirmed using XRD analysis, and it was confirmed that the CC-AgNPs were a face-centered cubic (fcc) crystalline-structured material. Furthermore, the role of active functional groups involved in the reduction and surface capping of CC-AgNPs was revealed using the Fourier transform infrared (FTIR) spectroscopic technique. CC-AgNPs were mostly spherical and monodispersed, with an average size of 26.89 nm, and were shown to be stable for a longer period without any noticeable change at room temperature. Further, we checked the antifungal mechanism of CC-AgNPs against C. auris MRL6057. The minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) were 50.0 µg/mL and 100.0 µg/mL respectively. The cell count and viability assay confirmed the fungicidal potential of CC-AgNPs. Further, the analysis showed that CC-AgNPs could induce apoptosis and G2/M phase cell cycle arrest in C. auris MRL6057. Our results also suggest that the CC-AgNPs were responsible for the induction of mitochondrial toxicity. TUNEL assay results revealed that higher concentrations of CC-AgNPs could cause DNA fragmentation. Therefore, the present study suggested that CC-AgNPs hold the capacity for antifungal drug development against C. auris infections.In terms of reduced toxicity, the biologically inspired green synthesis of nanoparticles has emerged as a promising alternative to chemically fabricated nanoparticles. The use of a highly stable, biocompatible, and environmentally friendly aqueous extract of  Green nanotechnology is a fast-emerging science with potential applications in the pharmaceutical, healthcare, biomedical, and drug delivery fields ,2,3. It C. auris strains.The synthesis of metal nanoparticles involves various physical and chemical routes, which are quite expensive, require high energy, and have various toxicity issues associated with these approaches , therefoCandida auris have been spreading and widely reported in different parts of the globe [C. auris is identified as a risk in healthcare units and leads to outbreaks. Additionally, unlike other species of Candida, this pathogen can persist and flourish for a long time on both dry and moist surfaces in clinics and hospitals [C. auris was described [Cynara cardunculus extract assisted preparation of silver nanoparticles using a simple, nontoxic, and economical approach. The structural properties of CC-AgNPs were investigated using different spectroscopic and microscopic techniques to determine the surface morphology, elemental composition, crystallinity, and optical properties. Further, the present study aimed to investigate the antifungal activities of CC-AgNPs against C. auris strains.Silver nanoparticles synthesized using green chemistry approaches show antioxidant , antibache globe . This sphe globe  as an evospitals ,46. The escribed  and the escribed  and actiCynara cardunculus, commonly known as artichoke, was collected from a local market in Jeddah, Saudi Arabia. Silver nitrate  and ethanol  were purchased from Sigma–Aldrich, St. Louis, MO, USA. All the chemicals used in this study were of analytical grade and were used without additional treatment. Highly pure double-distilled water (DDW) was utilized for the preparation of silver precursor and Cynara cardunculus solution.Cynara cardunculus were washed several times with distilled water, dried until the moisture was completely removed, and then ground into a fine powder. The Cynara cardunculus powder (10 g) was dispersed in an Erlenmeyer flask containing 250 mL distilled water and further heated at 80 °C for 60 min to achieve the completed extraction of biomolecules. The unfiltered solution was kept at room temperature for 12 h, and after that, the resulting extract was filtered through Whatman filter paper No. 1 using vacuum filtration apparatus. The filtered aqueous solution of Cynara cardunculus was stored in a refrigerator at 4 °C for further experimental use. It is recommended that fresh Cynara cardunculus extract (no more than 5 days after extraction) is used for synthesizing CC-AgNPs.The collected Cynara cardunculus extract required for the synthesis of CC-AgNPs. After several optimizing experiments, 14 mL of Cynara cardunculus extract was added to 20 mL of 1.4 × 10−4 M silver nitrate solution under continuous stirring using a magnetic stirrer. The color of the reaction mixture changed after just 5 min of reaction time and was analyzed using a double-beam Thermo Scientific Evolution 300 UV–visible spectrophotometer. The Cynara-cardunculus-mediated CC-AgNPs were purified, and the precipitated pellets were collected by using a BIOBASE centrifuge at a centrifugation speed of 5000 rpm for 20 min. The acquired pellets were then dispersed in distilled water and successively washed several times to completely remove the unbound compounds from the surface of the CC-AgNPs. Furthermore, the obtained material was subsequently dried at 90 °C for 5 h and then calcined at 500 °C for 3 h in a muffle furnace to remove all surface impurities and increase the crystallinity.The preparation of the silver nanoparticles was initiated by optimizing the amount of Cynara cardunculus extract was initially validated by recording the absorbance of the reaction mixture in the wavelength range of 200–800 nm using a double-beam Thermo Scientific Evolution 300 UV–visible spectrophotometer . All spectra were recorded at room temperature, in a quartz cuvette cell (path length 1 cm). A powder X-ray diffractometer (XRD)  set to 40 kV and 40 mA with 1.54 Å CuKα radiation was used to acquire the XRD pattern of the as-prepared CC-AgNPs in the scan range of 20–80 θ. Fourier transform infrared spectroscopy (FTIR) analysis of CC-AgNPs was performed on a Bruker ALPHA II FT-IR  spectrometer to assess the possible involvement of the functional groups in the Cynara cardunculus extract in the reduction and stabilization/capping of the CC-AgNPs. Transmission electron microscopy (TEM)  measurements were performed to analyze the morphology and the particle size distribution of the CC-AgNPs. Scanning electron microscopy (SEM)  equipped with an energy dispersive spectroscopy (EDS) was used to investigate the surface morphology and the elemental composition of the CC-AgNPs. Malvern Zetasizer  examined the zeta potential and the particle size distribution of the CC-AgNPs. The thermal stability of the as-prepared CC-AgNPs was analyzed using thermogravimetric analysis (TGA) in the temperature range of 30–800 °C under a N2 atmosphere with a heating rate of 10 °C/min using a Perkin-Elmer Pyris Diamond thermogravimetric analyzer (PerkinElmer LAS (UK)Ltd., Llantrisant, UK).The successful reduction of the silver metal ions using C. auris clinical strain MRL6057 was used. The strain was obtained from the National Institute of Communicable Diseases (NICD), South Africa, and preserved in the department as a glycerol stock. The antifungal action of CC-AgNPs was evaluated against C. auris MRL6057 by using a broth microdilution assay recommended in the standard M27 document (fourth ed.) [In the present study, the rth ed.) . The conTM. Briefly, yeast cells were grown for 8–10 h in Sabouraud dextrose broth (SDB) (Merck (Pty) Ltd., Johannesburg, South Africa) followed by centrifugation  and resuspension in fresh growth media. The yeast cells were adjusted to a density of 1 × 106 CFU/mL and subjected to various strengths of test NPs  for 4 h. Afterward, the yeast cells were washed and mixed with a MuseTM kit reagent (20 µL of yeast cells + 380 µL reagent), then incubated for 5 min at room temperature. The viability and cell count were estimated using a MuseTM cell analyzer. The experiment included a negative and positive control .The candidacidal phenomenon of CC-AgNPs was quantified using the count and viability kit provided by Muse6 CFU/mL. Later, the cells were subjected to various strengths of CC-AgNPs  for 4 h. In post-incubation, the cells were washed and fixed in chilled 70% ethyl alcohol , mixed with cell cycle reagent in equal proportions, and incubated in the dark for 30 min. The experiment included both negative and positive control.The impact of NPs on the yeast cell cycle was investigated using a Muse™ cell cycle kit following the steps given by the manufacturer. Briefly, the cells were propagated for 8–10 h in a fresh medium (SDB) and then centrifuged at 3000 rpm for 4 min. Then, the cells were re-suspended in a fresh medium and the turbidity was adjusted to 1 × 10C. auris was measured using a JC-10 assay kit . The experiment was done using the steps given by the manufacturer. The cells (mid-log phase) were subjected to various concentrations of test NPs  for 4 h. They were subjected to protoplast preparation, as described previously by Lone et al. [C. auris protoplasts were mixed with 50 µL JC-10 dye and distributed in different wells of a 96-well microtiter plate  for 1 h in the dark. After that, 50 μL of buffer-B was added to the plate and centrifuged for 2 min at 800 rpm. The readings were captured at Ex/Em = 490/530 nm (X) and 540/590 nm (Y) in microplate readers . The variation was measured in terms of the Y/X ratio. A decreased ratio confirmed the depolarization of the mitochondrial membrane. Moreover, the experiment included both negative and positive control.The impact of test NPs on the mitochondrial membrane potential of e et al. . Then, 9C. auris MRL6057 due to CC-AgNPs were examined using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The protocol used the In Situ Cell Death Detection Kit, Fluorescein , and the instructions provided by the manufacturer were followed. Briefly, C. auris cells were subjected to various concentrations of NPs  for 4 h and were subjected to protoplast preparation. Later, Triton X-100 (0.25%) was used to permeabilize the protoplasts, followed by incubation at 37 °C for 20 min. Later, the cells were mixed with TUNEL reagent and incubated at 37 °C for 1 h in a dark humidified box. Subsequently, samples were examined with fluorescence microscopy at Ex/Em = 495/519 nm . The experiment included both negative and positive control.The DNA fragmentation and condensation in The cytotoxic potential of various concentrations of given NPs  was evaluated on horse RBCs  and stated as percent hemolysis. The method was adopted from a method reported elsewhere . One perp-values ≤ 0.05 were measured statistically.Experiments were repeated thrice, and a two-way ANOVA test was used for determining the statistical significance of the results. Additionally, Cynara cardunculus extract as a reducing and capping/stabilizing agent. The incorporation of Cynara cardunculus extract in silver nitrate aqueous solution resulted in a gradual color change of the reaction mixture from pale yellow to brown and finally to a deep brown, which implied the formation of stable CC-AgNPs. The existing water-soluble polyphenolic molecules in Cynara cardunculus extract successfully reduced the available silver metal ion (Ag+) to metallic silver nanoparticles (Ag0) [Cynara cardunculus extract metabolites similarly play the role of surface-capping agents to prevent the nanoparticles from agglomerating [Cynara cardunculus extract is depicted in systematic The green and sustainable fabrication of metal nanomaterials using polyphenolic compounds has attracted huge attention because it has more advantages over the chemical route synthesis of nanomaterials ,52. Herees (Ag0) . The bioes (Ag0) . Finallymerating . The proCynara cardunculus, commonly named the artichoke plant, originated in southern Europe, and belongs to the family Asteraceae, which includes daisies and sunflowers, and is mostly cultivated as a horticultural crop in Italy [Cynara cardunculus extract, mostly concentrated in the roots, including hydroxy-octadecatrienoic acid, hydroxy-oxo-octadecatrienoic acid, tyrosyl-l-leucin, and dihydroxy-octadecatrienoic acid [in Italy ,56,57. Tin Italy ,59 and nin Italy ,61; (ii)in Italy ; (iii) sin Italy ; (v) trioic acid .Cynara-cardunculus-extract-mediated CC-AgNPs. In general, UV-vis spectra were analyzed to infer valuable information about the shape, size, and distribution of the biosynthesized nanoparticles established using surface plasmon resonance (SPR) bands [Cynara cardunculus extract confirmed the biosynthesis of polyphenol capped AgNPs. Meanwhile, with the addition of Cynara cardunculus extract to colorless silver nitrate solution, the color of the reaction mixture changed from light yellow to brown within 5 to 10 min of incubation. Furthermore, the reaction mixture turned dark brown after 30 min of incubation, demonstrating the reduction of silver metal ions (Ag+) to silver nanoparticles (Ag0). The Cynara cardunculus extract phytochemicals acted as reducing and stabilizing agents in the biogenic synthesis of stable CC-AgNPs. The present study involved further exploration using different AgNO3 concentrations and different volumes of Cynaracardunculus aqueous extract, and the stability of the as-prepared CC-AgNPs was also investigated by recording the UV-visible spectra at different times from 30 min to 360 min, as shown in 3, the increased absorption intensities were predominantly intensified. The fact of increased intensity with increased concentration of AgNO3 also emphasized the biogenesis of CC-AgNPs from the Cynara cardunculus extract increases. It is worth mentioning that the observed increase in the absorption intensity with the increase in time was due to the reduction of silver ions (Ag+) to elemental silver (Ag0). To determine the role of increased plant extract on the biosynthesis of CC-AgNPs, different plant extract concentrations ranging from 2 mL to 14 mL were explored under UV-vis spectra, as depicted in Cynara cardunculus extract. The study was further extended to establish the role of different time intervals ranging from 30 min–24 h under optimal reaction conditions, as depicted in Cynara cardunculus extract, and CC-AgNPs under optimal reaction conditions. Furthermore, the UV-visible spectra data revealed that the absorbance intensity of the reaction mixture increased with time, and the solution remained steady after more than 24 h of incubation, indicating that stable nanoparticle formation in the solution was successfully completed [The UV-visible spectra were obtained as the initial analysis to record the formation of R) bands . The UV-R) bands . The plaR) bands . The posR) bands ,66,67,68ompleted ,70. The Cynara cardunculus extract as reducing/stabilizing agents in the biogenesis of CC-AgNPs, as depicted in Cynara cardunculus extract to analyze the biosynthesis of CC-AgNPs. −1, 2860.8 cm−1, 1616.6 cm−1, 1489.7 cm−1, 1261.1 cm−1, 1218.8 cm−1, 1187.8 cm−1, 1080.6 cm−1, 998.7 cm−1, 632.0 cm−1, and 530.4 cm−1. However, the Cynara cardunculus extract possessed similar peaks as observed in standard CC-AgNPs, which demonstrated the biogenesis of CC-AgNPs upon phytochemical reduction. The peak intensity band that appeared at approximately 3300 cm−1, i.e., 3312.2 cm−1 and 3298.1 cm−1, were attributed to the presence of polyphenolic or polysaccharide –OH stretching vibrations. However, it was reported that the enol forms of such polyphenols or polysaccharides are reduced to quinone in extract mixture and are usually interpreted as a peak shift of –OH groups toward a higher frequency ranging between 3400 cm−1 and 3456 cm−1 [−1, which was assigned to C–H typical stretching vibrations from the CH2 groups of aliphatic compounds and are believed to have occurred after the reduction of AgNO3 [−1 and 2760 cm−1, i.e., 2790.3 cm−1, in the Cynara cardunculus extract was attributed to the presence of N-CH3 and C-H stretching vibrations corresponding to methyl-amino substituted groups [−1 and 1616.6 cm−1 lying between 1650 cm−1 and 1600 cm−1 corresponded to stretching vibrations due to conjugated ketones and aromatic ring stretching (-C=C-C) vibrations. The presence of peaks between 1550 cm−1 and 1400 cm−1, for example, 1534.8 cm−1 and 1489.7 cm−1, is believed to be due to the N-O stretching vibration of aromatic nitro compounds. The existence of an intensity peak in the range between 1410 cm−1 and 1310 cm−1, such as the 1388.1 cm−1 peak, in the Cynara cardunculus extract was assigned to –OH bending in phenolic and tertiary alcoholic groups. Similarly, the peak at 1252.7 cm−1 in Cynara cardunculus extract and the peak at 1261.1 cm−1 in CC-AgNPs were due to the presence of C-N stretching vibrations of primary aromatic amines. In addition, the occurrence of sharp peak intensities at 1083.4 cm−1 and 1080.6 cm−1 were the corresponding C-N stretching vibration bands of aliphatic amines, in the Cynara cardunculus extract and CC-AgNPs, respectively. Moreover, the peaks between 1320 cm−1 and 1210 cm−1, viz., 1227.3 cm−1 of the Cynara cardunculus extract and 1218.8 cm−1 of the CC-AgNPs, were attributed to C-O stretching vibrations. Furthermore, the peaks between 1190 cm−1 and 1130 cm−1, i.e., 1185.0 cm−1 of the Cynara cardunculus L. extract and 1187.8 cm−1 of the CC-AgNPs, signified the presence of C-N stretching vibrations of secondary amines. The observed sharp intensity peak in the range 1055 cm−1–1000 cm−1, i.e., 1007.2 cm−1 of the Cynara cardunculus extract, was due to the corresponding cyclohexane ring vibrations. The observed peak intensities at relatively lower frequencies between 850 cm−1 to 1000 cm−1, i.e., 900.0 cm−1 of the Cynara cardunculus extract and 998.7 cm-1 of the CC-AgNPs, were attributed to the hydrogen-bonded OH out-of-plane bending vibration modes. Meanwhile, the observed peaks between 660 cm−1 and 630 cm−1, such as 637.6 cm−1 of the Cynara cardunculus L. extract and 632.0 cm−1 of the CC-AgNPs, were believed to occur due to the presence of C-S thio-substituted compounds. The important observed peak intensity in both Cynara cardunculus L. extract and CC-AgNPs at 530.4 cm−1 was caused by the reduction of Ag+ to Ag0 in the biosynthesis of CC-AgNPs from Cynara cardunculus aqueous extract. In conclusion, the presence of polyphenolic vibrations, along with other functional group stretching vibrations, of Cynara cardunculus extract demonstrated their role as reducing and stabilizing agents in the biosynthesis of CC-AgNPs.The FTIR analysis was used to deduce the role of different functional groups present in the phytochemical composition of 456 cm−1 . A protrof AgNO3 . The exid groups . In addiCynara cardunculus extract was approximately 26.64% by weight. This observed weight loss in the first region between 0–220 °C was 1.81% by weight occurred after the loss of the adsorbed surrounding moisture and volatile residues of phytochemicals onto the surface of CC-AgNPs. Furthermore, the observed TGA peak at 63 °C for the derivative weight (by %) of −0.025 corresponded to the degradation of volatile phytocompounds onto the surface of the biosynthesized CC-AgNPs. The second weight loss between 150–300 °C was 12.6% by weight, which was observed after the thermal degradation of heterocyclic volatile phytochemical compounds that thermally decomposed onto the adsorbed surface of CC-AgNPs. The third region of thermal decomposition after 300 °C, with the DTG peak at 320 °C for the derivative weight (by %) of −0.093 and the DTG peak at 564 °C for the derivative weight (by %) of −0.071 were attributed to the thermal decomposition with a weight loss (by % weight) of about 12.23%, which was believed to occur after the thermal decomposition of phytochemical constituents, including polyphenolic compounds, flavonoids, and polysaccharides. These phytocompounds have an important appealing role in the capping and stabilization of the biosynthesized surface morphology of CC-AgNPs. The overall results of the TGA/DTG demonstrated the thermally stable biosynthesis of CC-AgNPs from Cynara cardunculus extract.The phytochemicals play essential roles as stabilizing and capping agents in the biogenesis of nanoparticles. Thus, the weight loss and thermal stability of such were investigated, which depended upon the adsorption of phytochemicals onto the surface of biogenic CC-AgNPs. Although the thermogravimetric analysis was operated at a heating rate of 10 °C/min under a nitrogen atmosphere, we inferred the weight loss in a stepwise manner for biogenic CC-AgNPs, as depicted in Cynara cardunculus extract was obtained and is depicted in Cynara cardunculus extract.The as-synthesized CC-AgNPs were subjected to XRD analysis to deduce the crystalline nature of the particles. The XRD pattern of the green-synthesized CC-AgNPs from Cynara cardunculus aqueous extract showed them to be well defined with a homogenous distribution of nearly spherical nanoparticles with an average particle size ranging between 14 to 43 nm. The larger particle sizes were observed due to the formation of agglomerates of the small particles. The average (23.74 nm), minimum (9.07 nm), and maximum (59.28 nm) particle sizes of the as-prepared CC-AgNPs were found from the particle size histogram using ImageJ software, as shown in Cynara cardunculus aqueous extract were of high purity.The transmission electron microscopy (TEM) and scanning electron microscopy–energy-dispersive X-ray spectroscopy (SEM-EDX) analysis of the as-synthesized CC-AgNPs were used to perform the study of the exterior surface morphology, structural characterization, and elemental composition, as shown in Cynara cardunculus extract in their colloidal state. The results obtained using zeta potential analysis are depicted in A zeta potential analysis was undertaken to analyze the stability of the biosynthesized CC-AgNPs from C. auris MRL6057, and the MIC values were reported as 50.0 µg/mL, whereas the MFC was found to be 100.0 µg/mL. In comparison, the MIC and MFC values of AmB against C. auris MRL6057 were found to be 4.0 and 8.0 µg/mL, respectively. According to published MIC breakpoints for C. auris [C. auris with MIC ≥2 µg/mL are considered resistant to AmB. Therefore, C. auris MRL6057 was deemed to be resistant to AmB.The CC-AgNPs were found to be active against C. auris , the cliC. auris displays high resistance to commonly used drugs [C. auris isolates to fluconazole [C. auris were found to be resistant to voriconazole and AmB, respectively [C. auris and their ability to persist in healthcare settings has redrawn the attention of researchers and healthcare experts to this pathogenic yeast [ed drugs . In vitrconazole . In compectively ,79. Furtectively . The gloC. albicans, and AgNPs were found to be potential inhibitors of growth and viability, both alone and in combination; for instance, a low strength of 1.8 mg/mL AgNPs in combination to cationic carboxilane inhibited the growth of C. albicans [C. auris. Therefore, further research that analyzed the in-depth mechanism of antifungal action of CC-AgNPs was required.Researchers investigated the candidacidal potential of AgNPs against albicans . Additioalbicans . These fC. auris was performed against CC-AgNPs to measure the growth and viability of these cells , the degree of DNA fragmentation was not much, which was depicted by fewer TUNEL-positive yeast cells. However, with increasing concentration (50–100 μg/mL), the degree of DNA damage also increased, which was reflected in the higher number of TUNEL-positive yeast cells.The microscopic analysis for the TUNEL assay results revealed that at higher concentrations of CC-AgNPs, green solid fluorescence suggested DNA breakage, as observed for cells treated with 10 mM H2O2 . TherefoC. auris and other pathogenic yeasts. It was also predicted that these nanoparticles, besides damaging nucleic acids in C. auris, can also damage antioxidant enzymes and cause lipid peroxidation in fungal cells. Overall, the results from this study suggested that CC-AgNPs led to nucleic acid fragmentation and mitochondrial membrane depolarization, which are the characteristic markers of apoptosis, thus, validating the idea that AgNPs induce late apoptosis in yeast cells and have dual antifungal action modes, including membrane disruption. The overall result suggested that CC-AgNPs led to mitochondrial membrane depolarization and DNA fragmentation, which are crucial apoptosis characteristics.DNA fragmentation is one of the significant markers related to morphological changes to identify the late apoptosis in yeast cells. The DNA fragmentation can be visualized using a TUNEL assay, which labels the free 3′-OH termini with modified nucleotides catalyzed by terminal deoxynucleotidyl transferase. The TUNEL assay was observed as the most dependable method to study late apoptosis . The tesThe CC-AgNPs in this study showed potent antifungal activity with a dual antifungal mode of action by causing cell cycle arrest and cellular apoptosis. However, to further escalate these nanoparticles to the next steps of drug development, it is essential to check their toxicity on host cells. To this end, the CC-AgNPs were tested for hemolytic activity against horse blood cells. The results obtained in this study revealed only 3–7% cell lysis when treated with CC-AgNPs at ½ half of MIC and MIC respectively. Cynara cardunculus extract as a reducing and capping agent. The present work was facile, cost-effective, and ecofriendly and did not require any solvent except water, which made this process highly advantageous. The formation of CC-AgNPs was confirmed by various microscopic and spectroscopic techniques before utilizing them for antifungal activities against C. auris. The experimental conditions for the preparation of CC-AgNPs were optimized via a surface plasmon (SPR) peak at 438 nm using UV-visible spectroscopy. Furthermore, the CC-AgNPs directly inhibited the cell cycle and arrested cells in the G2/M phase and could be a potential lead for antifungal drug development. Our results demonstrated that the as-prepared silver nanoparticles had good antifungal performance against C. auris and could be further explored for exceptional and enhanced biomedical applications.In this work, chemically stable silver nanoparticles (CC-AgNPs) were prepared via a phytochemically induced synthesis process using


Although costimulatory molecules have been shown to boost antitumor immune responses, their significance in stomach adenocarcinoma (STAD) remains unknown. The purpose of this study was to examine the gene expression patterns of costimulatory molecule genes in patients with STAD and develop a predictive signature to aid in therapy selection and outcome prediction. We used 60 costimulatory family genes from prior research to conduct the first complete costimulatory molecular analysis in patients with STAD. In the two study groups, consensus clustering analysis based on these 60 genes indicated unique distribution patterns and prognostic differences. Using the least absolute shrinkage and selection operator and Cox regression analysis, we identified nine costimulatory molecular gene pairs (CMGPs) with prognostic value. With these nine CMGPs, we were able to develop a costimulatory molecule-related prognostic signature that performed well in an external dataset. For the patients with STAD, the signature was proven to be a risk factor independent of the clinical characteristics, indicating that this signature may be employed in conjunction with clinical considerations. A further connection between the signature and immunotherapy response was discovered. The patients with high mutation rates, an abundance of infiltrating immune cells, and an immunosuppressive milieu were classified as high-risk patients. It is possible that these high-risk patients have a better prognosis for immunotherapy since they have higher cytolytic activity scores and immunophenoscores of CTLA4 and PD-L1/PD-L2 blockers. Therefore, our signature may help clinicians in assessing patient prognosis and developing treatment plans. Gastric cancer (GC) is a global public health burden, affecting more than one million individuals and causing an estimated 769,000 deaths  each year . Owing tMultiple clinical studies are combining immune checkpoint inhibition (ICI) therapy with conventional chemotherapy. Success has been documented in non-small- and small-cell lung carcinomas , 7, as wIn this study, we examined the expression patterns and prognostic significance of costimulatory molecular gene pairs (CMGPs) in patients with STAD. We then created and verified a predictive signature and nomogram for these patients. In STAD, a risk model based on CMGPs showed promise in predicting survival. Furthermore, a nomogram combining a risk model with clinical parameters effectively predicted the prognosis of patients with STAD. Finally, we assessed the potential response to immunotherapy and chemotherapy among several patient groups classified using the CMGP-based signature. Notably, similar data mining, processing, and model building have been achieved in renal cell carcinoma , 22, prohttps://portal.gdc.cancer.gov/), and the dataset was randomly divided into a training cohort and an internal test cohort at a 7:3 ratio. Furthermore, we used data from the Gene Expression Omnibus (GEO) (GEO-GSE15459) database (https://www.ncbi.nlm.nih.gov/geo) as the external validation cohort. In tumor immunotherapy, the tumor immune checkpoint pathways PD-L1/PD-1 and CD86/CTLA4 belong to the B7-CD28 family, and other costimulatory pathways mainly originate from the tumor necrosis factor (TNF) family. At present, 13 molecules are classified as members of the B7-CD28 family, including 8 molecules belonging to the B7 family  and 5 molecules belonging to the CD28 family . The TNF family consists of the TNF ligand superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF) with 47 molecules. Among them, 18 ligands are members of the TNFSF, and the other 29 receptors are members of the TNFRSF. Herein, we identified 60 costimulatory molecule genes (CMGs) from the study by Zhang et al. . The least absolute shrinkage and selection operator (LASSO)–Cox regression model was built using the prognostic-associated CMGPs derived from the univariate Cox regression analysis. We then used the LASSO method with penalty parameter tweaking, conducted via 10-fold cross-validation, to exclude the CMGPs that may be substantially associated with other CMGPs. A subset of CMGPs was identified by decreasing the regression coefficient with a penalty proportional to their size. For future multivariate Cox regression analysis, the CMGPs with nonzero regression coefficients were maintained. We compared the predictive CMGP values with the regression coefficients from the multivariate Cox proportional hazard regression analysis (β) to create a risk score model. For the LASSO regression analysis of the prognostic CMGPs, the “glmnet”  were calculated using the “ESTIMATE” package , and genp < 0.05 as a threshold (FDR < 0.25).We used cp.kegg.v7.1.symbols.gmt and go.v7.4.symbols.gmt in the “cluster profiler” package  to analyWe used the “RMS”  R packaghttps://www.cancerrxgene.org/), and the “pRRophetic” package . The prp-value of the two-stage test is still less than 0.05, indicating that costimulatory molecular genes are indeed an important factor affecting survival, and the overall survival of C2 patients is longer than C1.The workflow of this study is illustrated in p < 0.001), estimated scores (p < 0.001), and stromal scores (p < 0.001) than those in cluster 1 (p = 1.9e−13) (+ T cells (p < 2.22e−16) (p < 2.22e−16) (p < 2.22e−16) (p = 3.8e−05) (p < 2.22e−16) (p < 2.22e−16) (p = 0.1) (p = 0.78) (p = 0.087)  were identified between the two clusters, and functional investigations were performed to investigate the underlying signaling processes. Cluster 2 had 893 DEGs, 126 of which were downregulated and 767 were upregulated, compared with cluster 1. To evaluate the relationship between the enriched pathways and the prognosis of the patients with STAD, we utilized GSVA analysis to analyze the relative expression differences in the pathways in the two clusters. The heatmap in We examined the distribution of the clinical characteristics in the two clusters. The analysis showed that there was a significant difference in grade and stage, but none in age, sex, or other clinical features (p < 0.001), as evidenced by the survival curve  (The prognosis of the high-risk group was considerably poorer than that of the low-risk group in the TCGA test cohort (n = 192) and complete clinical data. The risk score for each patient in the cohort was determined using a prior method, and the patients were classified into high- or low-risk groups based on the unified cutoff value (0.6378). The high-risk group had significantly lower OS rates than the low-risk group, consistent with earlier research findings . In the high- and low-risk groups, principal component analysis revealed different distribution patterns  and eosinophil infiltrates (p = 0.008) than the low-risk group; meanwhile, the low-risk group had significantly more CD8+ T cells (p < 0.001), CD4 memory-activated T cells (p < 0.001), follicular helper T cells (p = 0.002), and M1 macrophages (p < 0.001) than the high-risk group  (p = 5.9e05) (p = 0.047) had a better prognosis (p = 0.79) had no statistical significance. Pearson correlation analysis showed that the estimated score    was negatively correlated with the risk score  and the risk score. Accordingly, the risk score can be used to analyze the TME.The patients with a lower estimate score (p = 0.00022) between high- and low-risk groups  . The B7-CD28 family includes B7-H3 (CD276), an essential immunological checkpoint. B7-H3 is a protein produced by antigen-presenting cells and is involved in the suppression of T-cell activity. More importantly, it is overexpressed in a variety of human solid tumors and is often associated with poor prognosis among patients . The impWith the advancement of immunotherapy, it is critical to find biomarkers and select the most sensitive individuals to increase immunotherapy response rates. To investigate the overall prognostic value, we used a consensus clustering approach based on the 60 CMGs. According to the Kaplan–Meier curves, the patients in cluster 2 had a worse prognosis. These patients also had a high concentration of immune-related pathways, indicating that CMGPs are closely associated with the TIME. Patients classified under cluster 2 may have a poorer prognosis owing to immune system weakness or a limited immunological response.Risk profiles based on CMGs might provide fresh insights into the clinical care of patients with STAD. In colorectal cancer  and lungTo further determine the links between our signature and the TIME, we analyzed immune cell infiltration and tumor mutation patterns in the high- and low-risk groups. In our analysis, the high-risk patients exhibited a much greater immune cell infiltration than did the low-risk patients. In addition, we found that the number of immunosuppressive cells, such as gamma delta T cells, MDSCs, monocytes, immature dendritic cells, macrophages, plasmacytoid dendritic cells, T follicular helper cells, and regulatory T cells, was larger in the high-risk patients than in the low-risk patients, indicating the presence of an immunosuppressive microenvironment. Tumor cells use an immunosuppressive microenvironment to evade immune responses and accelerate disease development. Understanding the immunological microenvironment of each patient will help identify patients who are more likely to respond to immunotherapy and enhance treatment response rates when combined with innovative treatment options.+ T cells, thereby triggering tumor-targeted immune responses. Therefore, tumor gene mutations are considered the premise of antitumor immunotherapy (p < 0.001), suggesting that tTMB is strongly correlated with the efficacy of PD-1/PDL1 antibodies , Gene Expression Omnibus (GEO-GSE15459) database (http://www.ncbi.nlm. nih.gov/geo) and Genomics of Drug Sensitivity in Cancer (GDSC) (https://www.cancerrxgene.org/). The original contributions presented in the study are included in the article/The datasets generated during and/or analysed during the current study are available in The Cancer Genome Atlas (TCGA) database , the National-Level Innovation Training Program for Chinese College Students of the Ministry of Education of the People’s Republic of China  and the Research Level Improvement Project of Anhui Medical University .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Twenty-four percent of participants (10 cases) had a clinically significant decrease in SpO2 at the first assessment, compared to 30.1% (13 cases) at the second, with no significant difference. An ICC of 0.993 (95% CI: 0.987 to 0.996) was obtained for the total number of steps in the CST.Many people recovering from an acute episode of coronavirus disease (COVID-19) experience prolonged symptoms. Exercise testing is a feasible and cost-effective option for assessing exercise tolerance, fatigue, and dyspnea related to effort. Being that the Chester step test (CST) is a progressive, submaximal test for predicting aerobic capacity, it could be a good option to explore. This study aimed to determine the reproducibility of CST for assessing exertional desaturation and exercise capacity in patients post-COVID-19 disease. A cross-sectional study was conducted on post-COVID-19 patients. Two attempts of the CST were performed. The intraclass correlation coefficient (ICC) was used to assess agreement between the two tests. Forty-two symptomatic post-COVID-19 patients were included, the mean age was 53.8 ± 10.3 years, and 52% were female. There was no significant difference between both tests ( Many people recovering from an acute episode of COVID-19 experience prolonged symptoms, especially fatigue, dyspnea, and low exercise tolerance ,3,4. GivExercise testing is a feasible and cost-effective option for assessing exercise tolerance, fatigue, and dyspnea related to effort ,8. The sThe CST was designed initially to estimate exercise capacity in healthy participants but has also been used in patients with respiratory diseases ,11. HoweRegarding the learning effect, in a previous study in 83 healthy subjects, the CST was stable between retests, suggesting no learning effect . Being tA cross-sectional study was conducted on post-COVID-19 participants admitted for a follow-up program at the Hospital Virgen de la Torre  between March and May 2021. The inclusion criteria were as follows: (1) age ≥ 18 years; (2) diagnosis of COVID-19 by a positive polymerase chain reaction (PCR) assay for SARS-CoV2 in nasal and pharyngeal swab samples; (3) sequelae of COVID-19 disease and candidate for a pulmonary rehabilitation program  [2) and heart rate (HR) before and at the end of the test. A desaturation level greater than or equal to 4 points from rest and post-test < 90% was considered clinically significant [During the initial visit, demographic characteristics, underlying comorbidities, and history of hospitalization were collected by interview. Two attempts of the CST were performed on the same day, with 30 min of resting time between both, on a 20-cm step and the auditory stimuli set the pace of the test using the following sequence: first stage: 15 steps/min; second stage: 20 steps/min; third stage: 25 steps/min; fourth stage: 30 steps/min; and fifth stage: 35 steps/min . Each stnificant . The modnificant .t-test, the Mann–Whitney U-test, or the Chi-square test for normally distributed, non-parametric, or categorical variables, respectively. The intraclass correlation coefficient (ICC) was used to assess agreement between the two tests. The significance level was set at p < 0.05.All statistical analyses were performed using SPSS version 22.0 . The data distribution was assessed using the Shapiro–Wilk test and expressed as mean and standard deviation or median with percentile 25-percentile 75 (P25–P75) according to the distribution. Differences between each measurement were assessed using Student’s Forty-two symptomatic post-COVID-19 patients were included, the mean age was 53.8 ± 10.3 years, and 52% were female . The medp = 0.896). Twenty-four percent of participants (10 cases) had a clinically significant decrease in SpO2 at the first assessment , compared to 30.1% (13 cases) at the second, with no significant difference . An ICC of 0.993 (95% CI: 0.987 to 0.996) was obtained for the total number of steps in the CST. The decrease in SpO2 was −2.0 (−4.0–−1.0) points and −2.0 (−6.0–−1.0) in the first and second assessment, respectively, with no significant difference (p = 0.435). We found no differences between dyspnea and fatigue of the lower extremity at the beginning or at the end of the test.In the first test, the median (P25-P75) of the total number of steps in the CST was 123 (68–177), while in the second test, it was 129 (74–195). There was no significant difference between both tests  to improve their aerobic capacity, especially in those patients with persistent symptoms after COVID-19 pneumonia .1 46 ± 15% of pred) also showed excellent relative reliability  [Our results are in line with similar studies performed in other chronic respiratory diseases. Recently, in 66 patients with interstitial lung disease , an excellent intra-rater  and inter-rater reliability  were found for the CST . A simil97–0.99) . This fi97–0.99) .Although 6MWT is the most commonly test for assessing exercise capacity , is not Although the results showed that this test is an alternative to assess physical capacity and desaturation to exertion, it should be considered that this is probably not the case for all populations. For example, if people have musculoskeletal problems, especially in the knees, it might not adequately reflect physical ability. On the other hand, if people are obese, the load on the lower extremities when going up the steps is greater and may affect adequate performance during the test.2 was similar in both measurements, which provides relevant information on the ability of the CST to detect exertional desaturation. In addition, the rest time between two CST tests is unclear, so we followed the recommendations of clinical guidelines suggesting 30 min of rest between two tests in the 6MWT [The main limitation of this study was the lack of data regarding oxygen kinetics during the test; this information could not be recorded due to a lack of access to this equipment. However, the physiological response to the decrease of SpOthe 6MWT . TherefoThe CST is a reproducible test in patients recovered from an acute episode of COVID-19 who experience prolonged symptoms. Performing a single CST attempt may be sufficient to assess exercise capacity and exertional desaturation in this population. Therefore, CST may be used in the early stages of the evaluation of rehabilitation programs when there are economic or space constraints.


One participant who was initially seronegative in the control group remained seronegative after the booster dose. These findings suggest humoral immune response benefits of switching the standard immunosuppressive regimen to the regimen of mTORi, TAC, and prednisolone in KTRs during vaccination.Kidney transplant recipients (KTRs) have a suboptimal immune response to COVID-19 vaccination due to the effects of immunosuppression, mostly mycophenolic acid (MPA). This study investigated the benefits of switching from the standard immunosuppressive regimen (tacrolimus (TAC), MPA, and prednisolone) to a regimen of mammalian target of rapamycin inhibitor (mTORi), TAC and prednisolone two weeks pre- and two weeks post-BNT162b2 booster vaccination. A single-center, opened-label pilot study was conducted in KTRs, who received two doses of ChAdOx-1 and a single dose of BNT162b2. The participants were randomly assigned to continue the standard regimen  or switched to a sirolimus (an mTORi), TAC, and prednisolone  regimen two weeks before and two weeks after receiving a booster dose of BNT162b2. The anti-SARS-CoV-2 S antibody level after vaccination in the switching group was significantly greater than the control group (4051.0 [IQR 3142.0–6466.0] BAU/mL vs. 2081.0 [IQR 1077.0–3960.0] BAU/mL, respectively;  Coronavirus disease 2019 (COVID-19) has a high mortality rate among solid organ transplant recipients (SOTRs). The mortality rate has decreased over time among kidney transplant recipients (KTRs). The mortality rate during March 2020 to February 2021 was 34.6% among KTRs. However, after the widespread use of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines, the mortality rate among KTRs declined to 13.3% . A regimIn the present pilot study, we compared the immune response to the booster dose of BNT162b2 in KTRs who were on the standard immunosuppressive regimen and those who switched to the mTORi, low-dose TAC, and prednisolone regimen.This is a single-center, pilot, open-label study conducted at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand, from March to September 2022. Three months after receiving the BNT162b2 dose, we administered a booster dose (adapted from CDC recommendation ) of homo® or Advagraf®, Astellas, Tokyo, Japan) with a trough level of 4–7 ng/mL, MPA , and prednisolone. The immunosuppressive drugs in the switching group were mTORi  with trough level of 5–10 ng/mL, low-dose TAC  with trough level of 2–4 ng/mL, and prednisolone. The switching of the immunosuppressive regimen was started two weeks before the BNT162b2  booster was administered to restore the immune response which had been suppressed by MPA, and continued for two more weeks after the booster vaccination to allow the immune response to build up against COVID-19 . Sirolimus was administered as a loading dose using 6 mg in day 1 and henceforth followed by 2 mg daily. Low-dose tacrolimus was administered as a half dose of the previous dosage. After switching the drugs for 2 weeks, the trough levels of tacrolimus and sirolimus were measured to adjust the dosage of the drugs.The participants were enrolled into the study by the research assistant using a computer. The enrolled participants were randomly assigned in a 1:1 ratio using a block of four randomization to either continuing the standard immunosuppressive regimen (control group) or switching to the mTORi, TAC, and prednisolone regimen (switching group). The randomization was performed by an independent statistician. Sequentially numbered, opaque and sealed envelopes were used for the allocation concealment. It is not feasible to conduct a double-blinded study because there is a distinction in the drug characteristics. The standard immunosuppressive regimen consisted of TAC . The study was conducted in compliance with the international guidelines for human research protection as per the Declaration of Helsinki and the International Conference on Harmonization in Good Clinical Practice (ICH-GCP). The study protocol was registered in the Thai clinical trial registration (TCTR20220404001). All participants provided written informed consent prior to their enrollment in the study and the medical records were thoroughly reviewed.®, by Cobas e 411 analyzer; Roche Diagnostics, Basel, Switzerland). The lower limit of detection was 0.36 binding antibody units (BAU)/mL. The anti-SARS-CoV-2 S antibody ≥0.823 BAU/mL was considered reactive or seroconverted. The adverse events were monitored for six months or until September 2022.Baseline characteristics and biochemical data of the participants were obtained from their electronic medical records. The mean of the previous three consecutive serum creatinine levels was used as the baseline serum creatinine for the participant. The outcome was a change in anti-SARS-CoV-2 S antibody level pre- and post-BNT162b2 vaccination. Blood samples were collected before vaccination and at 4 weeks after vaccination and centrifuged at 3000 rounds per minute for 10 min at room temperature and frozen at −20 °C until the anti-SARS-CoV-2 S antibody was measured .Continuous data were presented as mean ± standard deviation (SD) for Gaussian distributed data and median  for non-Gaussian distributed data. Categorical data were described as proportion and percentages. Baseline characteristics were compared between the groups using t-test for normal distributed data and the Wilcoxon rank-sum test for non-normal distributed data. The serum antibodies which were seronegative were imputed with 0.18 BAU/mL (50% of the lower limit of detection). The difference in the increment of the anti-SARS-CoV-2 S antibody level between the two groups was compared by t-test. The data were analyzed using Stata 14  and a p = 0.85), respectively. The duration since the last dose of BNT162b2 was comparable at approximately 5 months between both groups (p = 0.66). The white blood cells, neutrophil, and lymphocyte counts were also comparable between the two groups  and the switching group (14 participants). All participants completed the study . The meao groups . In the p = 0.61). After vaccination, the overall anti-SARS-CoV-2 S antibody level significantly increased to 3338.0 (IQR 2081.0–4983.5) BAU/mL (p < 0.001). The anti-SARS-CoV-2 S antibody level significantly increased in both groups (p = 0.001). The switching group had a significantly higher anti-SARS-CoV-2 S antibody level compared to the control group  (The median (IQR) baseline anti-SARS-CoV-2 S antibody level was 170.2 (IQR 36.0–510.3) BAU/mL, which was not different between the control group (204.9 [IQR 44.7–541.2] BAU/mL) and the switching group (164.5 [IQR 19.5–429.5] BAU/mL) ( = 0.01) .In this study, there was only one seronegative participant which was in the control group. The participant did not achieve seroconversion after the fourth vaccination.p = 0.87 in control group and 1.30 ± 0.48 vs. 1.32 ± 0.43 mg/dL; p = 0.50 in switching group) and urine protein  before and after the study, respectively.Two participants in the switching group developed mild oral ulcers that spontaneously resolved after changing the mTORi back to MPA at the end of the study. There were no other adverse events related to mTORi, such as edema and pneumonitis. In the control group, there were no serious local or systemic adverse events such as bleeding, bruises, chest discomfort, severe headache, vomiting, seizure, or stroke-like symptoms after vaccination . There wThe results in the present study indicated that there was a higher immune response after the booster dose of COVID-19 vaccine in the group that received the MPA-sparing regimen with mTORi and low-dose TAC compared to the standard immunosuppressive regimen. The only one seronegative KTR who was in the control group remained seronegative after the fourth COVID-19 vaccination.Many studies reported that SOTRs have lower immunological response to vaccination compared to the healthy population ,19,20,21The regimen of TAC, MPA and prednisolone is the standard immunosuppressive regimen widely used in kidney transplantation. However, the regimen which contained MPA provided lower immune response to many vaccines, such as influenza , hepatitIn a subgroup vaccination study of an RCT by Boer et al., conducted in 32 patients (16 patients on TAC [5 to 8 ng/mL] + MMF [1000 mg/day] + prednisolone and 16 patients on low dose TAC [1.5 to 4 ng/mL] + everolimus [3 to 6 ng/mL] + prednisolone), it was revealed that the SARS-CoV-2 anti-spike receptor binding domain IgG antibody levels were significantly higher in the low dose TAC + everolimus + prednisolone group after receiving two doses of mRNA vaccine . All patAfter the participants received the booster dose, the antibody level in the switching group was higher than those on the standard immunosuppressive regimen. This can be explained by two assumptions. Firstly, the cessation of MPA and use of low dose of TAC resulted in an improvement of the immune response. Secondly, mTORi may also augment the immune response. The poor immune response after vaccination in patients who were on MPA could be explained by MPA inhibiting IL-4+ CD4 T-cell and B-cell function, plasma cell formation, and antibody production ,27. ThusIn this study, a four-week period of switching to an MPA-sparing immunosuppressive regimen (two weeks before and two weeks after vaccination) was sufficient enough to improve antibody response. A recent study of MPA dose pause for one week before and four weeks after vaccination also revealed improvement in serologic response . In anotThe immune response to vaccination can be affected by various factors, such as the previous vaccination regimen and the duration after the last dose. The regimen of vaccination of all participants prior to entering this study was tightly controlled to be homogeneous, which was two doses of AZD1222 and a single dose of BNT162b2. Furthermore, the duration since the last vaccination was comparable between the two groups. Admittedly, this study only measured the anti-SARS-CoV-2 S antibody level. In this respect, it has been demonstrated that anti-SARS-CoV-2 S antibody level together, with neutralizing antibodies, could prevent the acquisition of SARS-CoV-2 infection and had a good correlation ,34. HoweImmunosuppressive drug switching from TAC, MPA, and prednisolone to the regimen of mTORi, TAC and prednisolone for two weeks before and two weeks after booster dose of COVID-19 vaccination can improve humoral response in stable KTRs.


Listeria monocytogenes is an opportunistic intracellular pathogen causing an infection termed listeriosis. Despite the low incidence of listeriosis, the high mortality rate in individuals at risk makes this bacterium one of the most dangerous foodborne pathogens. Reports about a relapse of infection after antibiotic treatment suggest that the bacteria may be able to evade antibiotic treatment and persist as a dormant, antibiotic-tolerant subpopulation. In this study, we observed intracellular generation of antibiotic-resistant L-forms of Listeria monocytogenes following Ampicillin treatment of Listeria monocytogenes infected cells. Detection and identification of intracellular Listeria L-forms was performed by a combination of fluorescence in-situ hybridization and confocal laser scanning microscopy. Using micromanipulation, it was possible to isolate single intracellular L-form cells that following transfer into fresh medium gave rise to pure cultures. In conclusion, the results obtained here provide strong evidence that antibiotic treatment of infected host cells can induce the formation of L-forms from intracellular Listeria monocytogenes. Furthermore, our results suggest that intracellular L-forms persist inside host cells and that they represent viable bacteria, which are still able to grow and proliferate. Listeria monocytogenes is a rod-shaped, non-spore forming, Gram-positive bacterium. This facultative intracellular pathogen causes listeriosis, a severe disease with a mortality rate of up to 30% in high-risk individuals comprising of young children, elderly people, pregnant women and immunocompromised individuals  and phospholipases are activated by the low pH inside the phagolysosome and allow the bacteria to escape into the cytoplasm. After the release into the host cell cytoplasm, L. monocytogenes can multiply and spread to neighbouring cells, driven by ActA-mediated polymerization of host-cell actin  strains and the walled, revertant L-form variants were grown in liquid Brain-Heart Infusion (BHI) broth  at 37°C with shaking. Listeria L-forms were grown at 37°C in liquid DM3 medium, which is an osmoprotective medium originally described by Strains used in this study are listed in Listeria monocytogenes that are readily able to switch between the cell wall-deficient L-form and the walled parental state through alteration of culture conditions. To generate Rev strains, 1:10, 1:100 and 1:1000 dilutions of overnight cultures of Listeria were inoculated in DM3 soft agar tubes containing 0.8%, 0.5%, or 0.2% agar  using a sterile inoculation needle or by dilution of an aliquot of overnight culture directly in liquid DM3. All media were supplemented with 200 μg/ml Penicillin G . After several days of incubation, newly formed colonies were successively inoculated into a fresh DM3 culture tube containing lower agar concentrations, until the L-form cells were adapted to grow in liquid DM3 medium. Subsequently, 1:10, 1:100 and 1:1000 dilutions of the L-form cultures were plated on solid DM3 agar medium plates, to support reversion of L-forms to the walled state  strains are variants of ed state . To veri® CCL-98™, passages 195 to 207) was cultured in F-12K Medium (ATCC® 30-2004) supplemented with 10% FBS . Human Brain Microvascular Endothelial Cells (HBMEC), passages 3 to 20, were cultured in RPMI-1640 medium with L-glutamine  supplemented with 1mM Na-Pyruvate (Sigma-Aldrich), 1% MEM non-essential amino acids (Sigma-Aldrich), 1% MEM Vitamin Solution x100 (Sigma-Aldrich), 10% Nu-Serum  and 10% FBS , adapted from 2 culture flasks  at 37°C and 5% CO2 in a humid atmosphere. To maintain the culture monolayer cell cultures were split when 70 to 90% confluence was reached.The placental choriocarcinoma cell line BeWo  were determined for both types of plates. The L-form conversion rate was calculated as the percentage of the ratio between the CFU for L-forms and the CFU for walled, parental cells. For this purpose, three biological replicates were performed.In Vitro Life Science Laboratories, Slovak Republic) for microscopic observation, at a density of 3x104 cells/cm2. Invasion assays were performed as previously described  of 10. An aliquot was plated in parallel on BHI agar to determine the number of bacteria (CFU/ml) added to the cell cultures. The culture vessels were incubated for 1 h at 37°C and 5% CO2. To stop the infection, the cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS)  and incubated for 60 min in full medium supplemented with 50 µg/ml of gentamicin to kill extracellular bacteria. For determination of CFU from intracellular bacteria, cells were lysed after 2 h using a solution of 0.5 M Succinate  in DPBS supplemented with 0.01% Saponin . The lysates were plated on BHI and DM3 agar plates and incubated over night at 37°C. For generation of intracellular L-forms, Ampicillin was added to the cell cultures 7 hours post-infection. Cell culture medium was renewed every two days, and fresh Ampicillin was added every day during the whole period of the experiment. During the initial phase of the cell culture experiments we used 200 µg/ml Ampicillin, because this concentration was used for the generation of revertant Listeria strains. Later, it became obvious that the Ampicillin concentration had an impact on the emergence of L-forms in vitro, and that concentrations between 25 µg/ml and 50 µg/ml were superior to generate intracellular L-forms.Prior to an experiment, BeWo and HBME cells were grown to 80% confluency. The cells were seeded into 6-well plates  for cell lysis, and 2-well Ibidi µ-Slides  or 50 mm Dish (MatTek escribed . All medin-situ hybridization (FISH) was first tested and established using pure L-form cultures. The bacteria were gently washed three times with a solution of 0.5 M Succinate  in DPBS. Fixation was performed with 0.25% Glutaraldehyde  for 15 min. After fixation, the sample was washed again three times and incubated for 30 min in 1 mg/ml NaBH4  to reduce the autofluorescence caused by the fixative. After another washing step, the samples were dehydrated by incubation for 3 min in increasing concentrations of ethanol: 50%, 80% and 100%, and thus air-dried. Listeria genus-specific probes Lis-637  were applied individually or in combination in the hybridization buffer . Hybridization was performed for 2 h at 46°C. The reaction was stopped by washing for 5 min at 46°C in pre-warmed wash solution . Samples were subsequently washed in distilled water, air-dried, and mounted in Citifluor AF1  to reduce photobleaching of fluorescent dyes. An almost identical protocol was used for infected cell cultures to detect the possible presence of Listeria L-forms inside eukaryotic cells, except that hybridization was performed overnight.Fluorescence  Lis-637  and Lis- Lis-637  were appe EUB338  was usede EUB338  and labeListeria were excited at 488 nm from a solid-state laser with a power of 1 mW at 100% AOTF . For imaging, the laser intensity was adjusted to 13% AOTF (130 μW), and signals were recorded using a PMT detector with a detection range of 510-550 nm. Microscopy of cells and cultures was performed at 37°C in a temperature-controlled chamber  to maintain optimal conditions for eukaryotic cell lines. For microscopy of FISH samples, the probe EUB338-ATTO488 was excited at 488 nm and the emission signal was collected in a detection range of 510-530 nm. Probes NON338-ATTO532, Lis-637-ATTO532 and Lis-1255-ATTO532 were excited at 532 nm, and the emission signal was collected in a range of 545-560 nm. Hoechst 33258 staining of eukaryotic cell nuclei was excited at 405 nm, and emission recorded in a range of 450-500 nm.Microscopic inspection of FISH samples was performed using a Leica TCS SPE inverted laser scanning confocal microscope  equipped with ACS APO 63x/1.30 OIL CS and a HCX PL FLUOTAR 100x/1.30 OIL PH3 objective lenses, unless stated differently. For live imaging, GFP-expressing Images shown in Images were acquired using the LAS AF software . Brightness, contrast, and alpha of images were adjusted using Fiji v2.1.0 (ImageJ software)  to produListeria L-forms was performed on a Leica TCS SPE inverted microscope equipped with the TransferMan® NK2 system and a CellTram® Air  as previously described by Isolation and transfer of single GFP-expressing Listeria L-forms from cell cultures was performed by following the same protocol at 37°C. For this purpose, BeWo and HBME cells were seeded in low, 50 mm Ibidi® µ-Dishes , to provide sufficient room for manipulation with the micro-pipette. Single intracellular L-form cells, or complete eukaryotic cells containing intracellular L-forms were extracted from the cell cultures using an ICSI blunt glass pipette with an inner diameter of 6 or 10 µm. The extracted Listeria L-form cells were transferred into fresh, pre-warmed antibiotic-free DM3 broth, and incubated at 37°C.The extraction of intracellular Listeria monocytogenes L-forms  was determined as 3.8 ± 1.3 days, which was much faster compared to the time needed for conversion. More than 90% of cells from Rev strains showed conversion to L-forms when cultivated overnight in DM3 containing antibiotics, in contrast to only 47% of the respective wild-type strains (n=40). Interestingly, the Rev strains did not show reversion in liquid DM3 without Penicillin G (tested for 3 to 5 passages), while rapid reversion could be observed on solid DM3 agar without antibiotics. This ability to readily switch between the parental and L-form state represented an important property and prerequisite for the investigation of the possibility of intracellular formation of L-forms upon antibiotic treatment.Listeria monocytogenes are able to convert to L-forms upon antibiotic treatment and possibly contribute to the persistence of Listeria monocytogenes inside host cells as a source for recurring infections, an in-vitro cell culture model was established. Human Brain Microvascular Endothelial Cells (HBMECs) were infected with the constitutively GFP-expressing revertant Listeria monocytogenes strain Rev10. The presence and fate of intracellular L. monocytogenes was assessed by confocal laser scanning microscopy. Seven hours post infection cultures were treated with Ampicillin. Two days later, typical rod-shaped parental Listeria were no longer visible in treated cell cultures. However, microscopic inspection of the cell cultures revealed the occurrence of spherical, green-fluorescent structures inside eukaryotic cells between days 2 and 14 after infection. These green-fluorescent, intracellular structures were subsequently identified as L. monocytogenes (Listeria monocytogenes.In order to investigate whether intracellular L. monocytogenes WSLC 1042 (serovar 4b) for infection of HBMEC cells, followed by an Ampicillin treatment and observation of intracellular L-form generation  approach using fluorescently labelled Listeria-specific probes. Because L-forms lack the rigid cell wall providing proper stability during the assay, the standard FISH protocol had to be adapted, and in-vitro assays with pure L-form cultures were used to demonstrate the suitability of the adapted protocol for L-form bacteria. The FISH results showed that fixation with Glutaradehyde was suitable and sufficient to preserve the shape of L-form cells. Strong fluorescence signals obtained from Listeria-specific probes Lis-637, Lis-1255, and universal probe EUB338, together with the absence of a signal from probe NON338, demonstrated specific labeling of Listeria L-form cells  strains with the property to readily switch between the L-form and the parental state, generated from two different serovars (1/2a and 4b). The obtained revertant strains demonstrate that the L-form state allows the bacteria to evade and survive antibiotic treatment. Furthermore, it shows that under certain conditions L-forms possess the capability to revert back to the parental state after the removal of the antibiotic. This finding is consistent with previous studies that have shown that different serovars of Listeria can be converted to L-forms, which retain the ability to revert back to their walled form  with fluorescent Listeria genus-specific oligonucleotide probes. Fluorescence in-situ hybridization has been previously applied to identify L-forms isolated from urine samples (Listeria L-forms. Because FISH is based on the hybridization of oligonucleotide probes to the ribosomal RNA, which is a substantial part of the ribosomes, it is broadly accepted that fluorescent signals will only be obtained from cells that have been alive and metabolically active at the time of fixation. In contrast, the fluorescence from GFP is not suitable as a viability marker since the protein remains detectable even in dead cells (Using cell culture assays and confocal laser scanning microscopy, we could demonstrate that Ampicillin treatment can trigger the intracellular generation of L-forms in host cells infected with GFP-expressing st cells  and thatst cells . Therefo samples  and showad cells . Howeverad cells , which aad cells . A possiListeria L-forms can persist inside macrophages for several weeks, thus corroborating the hypothesis that bacterial L-forms may play a role as intracellular persisters (Bacillus subtilis and Staphylococcus aureus, it was also reported that L-forms may occur in macrophages (Streptococcus faecalis L-forms back in 1974.In order to overcome the obstacles linked to the extraction of L-forms from cell cultures, a micromanipulator approach was established and applied for target-oriented extraction of intracellular L-form cells and their subsequent transfer into fresh osmoprotective DM3 medium. Using this approach, approx. 50% of the extracted L-forms showed growth after their transfer to fresh DM3 medium. This percentage was even comparable to the percentage obtained for the direct transfer of single L-form cells from a pure culture to fresh medium using a micromanipulator. These results indicate that the low transfer efficiency is not due to the intracellular location of the L-forms and a diminished capability of the L-forms to survive inside the host cells. In contrast, the low transfer efficiency may be explained by the metabolic heterogeneity of L-form populations, as some of the extracted L-forms might not have been metabolically active . Anotherrsisters . For Bacrophages  and GreeListeria monocytogenes to convert to L-forms in non-phagocytic cells after antibiotic treatment and that the pathogen may persist as viable L-forms inside the host cells. However, the intracellular reversion of Listeria monocytogenes L-forms to parental bacteria could not be observed, which was probably due to the low frequency of L-forms that developed and persisted inside the host cells and the restriction of the duration of monitoring the fate of intracellular L-forms as a result of the limited viability of host cells in cell cultures. Taken together, the results obtained in this study demonstrate that Ampicillin treatment can trigger the emergence of intracellular Listeria L-forms. Thus, these settings can be used for a more in-depth analysis of intracellular Listeria L-forms, as it is assumed that their behavior inside host cells is largely different from in-vitro culture (Listeria monocytogenes (Listeria ivanovii (The results from this study provide strong evidence for the ability of intracellular  culture . Furtherytogenes , but alsivanovii .Listeria monocytogenes L-forms can be induced by antibiotic treatment of infected cell cultures. Furthermore, L-forms can persist as viable cells that keep their property to grow and divide outside their host cells. Although the conditions under which L-form cells revert to their walled state are yet to be investigated, the current findings on the occurrence of intracellular Listeria L-forms support the hypothesis that they are likely to play a role in the persistence and resurgence of listeriosis and chronic infection. Thus, the evidence-based findings from this study suggest the need for an improved antibiotic treatment regime for listeriosis that may take into account the presence of intracellular cell wall-deficient L-forms of Listeria monocytogenes.In conclusion, the results from this study demonstrate that the development of intracellular The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.VG, MS, and ML conceived and designed the experiments. VG, CE, and IK performed the experiments. VG, IK, MS, and ML analyzed the data. VG prepared the original draft of the manuscript. MS and ML performed manuscript revision and editing. All authors contributed to the article and have read and approved the submitted version.This work was funded by the Swiss National Science Foundation SNF, project number 170042.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The COVID-19 pandemic caused changes to how healthcare services are utilised and delivered.We examine the impact of COVID-19 on the pattern of emergency patient presentations referred on to the community mental health team and the impact of utilising telemedicine on time to follow-up.We retrospectively reviewed all clinical records of patients currently attending our service. We identified presentations to the emergency department (N=119) who were subsequently referred on for mental health follow-up.Patients being referred to our team from emergency departments were significantly younger during, mean age 33.1 years (SD=12.3) compared to before the pandemic, mean age 40.0 years (SD=14.5), p=0.006 and a higher proportion were new patients during, 55.8%, compared to pre-pandemic period 33.3%, p=0.015. There was also a higher proportion of patients presenting with suicidal ideation and lower proportions of affective, psychosis and suicidal/self-injurious acts during the pandemic period compared to before, p=0.006. The ratio of female to male patients on the other hand were similar during both periods, p=0.853. There appeared to be no difference in median time to follow-up pre and during the pandemic . Further analysis also found no significant impact on time to follow-up upon implementing telemedicine consultations, with median days to initial follow-up of 6 days pre-pandemic, 4.5 days during pandemic + prior to telemedicine and 6.5 days during pandemic + telemedicine, p=0.602.This study provides preliminary data on the impact of COVID-19 on mental health emergency presentations and utilization of telemedicine on time to follow-up by CMHTs.No significant relationships.


Escherichia coli (ESBL E. coli) as an example of a resistant pathogen of concern. Is the short steaming procedure that blue mussels (Mytilus edulis) undergo before consumption enough for food safety in regard to such resistant pathogens? In this study, we performed experiments to assess the survival of ESBL E. coli in blue mussel. Consequently, a predictive model for the dose of ESBL E. coli that consumers would be exposed to, after preparing blue mussels or similar through the common practice of brief steaming until opening of the shells, was performed. The output of the model is the expected number of colony forming units per gram (cfu/g) of ESBL E. coli in a meal as a function of the duration and the temperature of steaming and the initial contamination. In these experiments, the heat tolerance of the ESBL-producing E. coli strain was indistinguishable from that of non-ESBL E. coli, and the heat treatments often practiced are likely to be insufficient to avoid exposure to viable ESBL E. coli. Steaming time (>3.5–4.0 min) is a better indicator than shell openness to avoid exposure to these ESBL or indicator E. coli strains.Pathways for exposure and dissemination of antimicrobial-resistant (AMR) bacteria are major public health issues. Filter-feeding shellfish concentrate bacteria from the environment and thus can also harbor extended-spectrum β-lactamase—producing  Shellfish such as blue mussels are consumed after a short heat treatment. As shellfish filter seawater, they accumulate microbes and the effectivity of the heat treatment for decontamination is therefore critical for food safety.Escherichia coli and Klebsiella pneumoniae, belonging to certain clonal lineages, has contributed to the rapid global spread of extended-spectrum beta lactamase-producing Gram negative bacteria (ESBLs) and carbapenemases [E. coli Sequence type (ST) 131 and K. pneumoniae ST258 as pertinent examples [E. coli is one of several emerging AMR microbes that have been detected in blue mussels [Antimicrobial-resistant (AMR) bacteria in seafood represent a potential risk for human beings by two main mechanisms, either clonal transfer of resistant bacteria or by horizontal gene transfer (HGT) of mobile genetic elements (MGEs) to previously susceptible pathogens. The emergence of successful multi-drug-resistant (MDR) variants of enemases . These c mussels ,5. The o mussels . The fil mussels . They th mussels ,5,7,8.E. coli to human beings is unknown. A study performed under the Norwegian monitoring program for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) in 2016 reported that 4.2% of E. coli isolates obtained from bivalve molluscs (n = 261) in Norway were resistant to at least one antibiotic, while the prevalence of resistance to three or more antibiotics was 1.4% [The potential for blue mussels to be a significant source of ESBL- producing was 1.4% . By usinwas 1.4% .E. coli/g by the end of manufacturing process for direct human consumption (Commission regulation (EC) No 2073/2005). Class A shellfish by harvest should not contain more than 230 cfu/g of E. coli by harvest. A recommended practice has been to move shellfish with higher E. coli concentrations to cleaner water until the concentration falls below this level or they will undergo an industrial heat treatment before being marketed [E. coli exposure may depend on their initial concentration and how well they survive heat treatment.Shellfish such as oysters and mussels are often consumed raw or undercooked for culinary reasons, which may pose a risk for the consumer . In addimarketed . WhetherE. coli and ESBL-producing E. coli and minimum heating conditions for elimination of these microbes in shellfish. There exists little knowledge about the persistence of viable ESBL-producing E. coli in different food matrixes where only light–heat treatments are performed before consumption, but both the maximum obtained temperature within the mussels as well as the duration of certain temperatures will likely have an impact on bacterial survival ratesIt is therefore a need for knowledge about the trade-off between safety and preferred sensory quality, i.e., the potential for survival of both E. coli in blue mussels following different heat treatment regimes, and to develop a corresponding exposure model and tool for risk assessment and guideline development. To achieve this, we conducted experiments inoculating live shellfish with E. coli as an indicator for ESBL-producing E. coli to avoid unacceptable contamination risks and used ESBL-producing E. coli in controlled heat treatment experiments comparing it to E. coli to verify their role as an indicator and accumulate additional data on heat inactivation.The aim of this study was therefore to assess the survival of ESBL-producing E. coli by allowing them to naturally filter contaminated water in an aquarium. This experiment could, however, not utilize ESBL strains due to biohazard procedures, and thus a second set of experiments addressed this by homogenizing a mix of mussel flesh and either non-ESBL or ESBL-producing E. coli in a series of heat-resistant plastic bags.The model for the mussel production chain developed in this work incorporates two sets of experiments. The first set of experiments involves inoculating live mussels with non-ESBL-producing Blue mussels for the study were purchased at a local supermarket, where they had been stored on ice. The mussels were transported to the lab and within 45 min placed at 5 °C or in the aquarium for acclimatization.Our approach consisted of two main steps:E. coli in an aquarium and then steaming them for different lengths of time in a kettle, thus closely simulating normal consumer pro-cedure and materials.  aths See .All experiments and analyses were conducted at the Norwegian Veterinary Insti-tute facilities in Oslo.E. coli  and ESBL-producing E. coli (ESBL)  were used. The five isolates were all isolated from blue mussels analyzed previously [For the inoculation studies described below, indicator eviously  and kindE. coli (2016-22-55-1-1-1-1) originating from a blue mussel purchased from a retail store was used for inoculation of the water in the aquarium experiment. Briefly, the inoculum was prepared from frozen glycerol stocks where a loopful (1 µL) was plated directly from the stock onto a blood agar plate (bovine blood) that was incubated at 37 ± 1 °C overnight. A single colony of E. coli from the blood agar was added to 100 mL Buffered Peptone Water  and incubated over night at 37 ± 1 °C. The overnight broth culture was equally distributed in four 50 mL sterile tubes and washed twice by centrifugation for 10 min at 3800 g (Beckman GS-15R Centrifuge), removal of the supernatant and resuspension of the bacterial pellet in 10 mL 0.9% saline water. After the second wash, the pellets were resuspended in 10 mL 0.9% saline separately prior to adding to the aquarium.One isolate of indicator E. coli (2016-01-4162-1-1-1-1 and 2016-01-4220-1-1-1-1) and two strains of ESBL-producing E. coli (2016-01-4162-1-3-1-1 and 2016-01-4220-1-3-1-1), respectively. Both ESBL-producing E. coli harbored blaCTX-M-15, where strain 2016-01-4162-1-3-1-1 harbored blaCTX-M-15 alone, while strain 2016-01-4220-1-3-1-1 also had blaCMY-2. These isolates originated from two samples in which both an indicator E. coli as well as an ESBL isolate had been detected through selective screening within the NORM-VET program. The inocula were prepared from frozen glycerol stocks by plating a loopful of stock on blood agar plates followed by incubation at 37 ± 1 °C overnight. A single colony from each isolate was transferred to separate tubes of 10 mL BPW-ISO and incubated as described above. After incubation, the two isolates of indicator E. coli and the two strains of ESBLs E. coli were mixed to an E. coli mix and an ESBL -E. coli mix, respectively, and used for direct inoculation of samples of blue mussel flesh.For the experimental inoculation of blue mussel flesh, samples were spiked with two isolates of indicator 8 pr mL E. coli (2016-22-55-1-1-1-1) was added. The aquarium experiment was carried out three times.Artificial seawater (3%) was made by adding 2100 g Red Sea Salt (© 2020 Red Sea) to approximately 58 L ice and cold 35 L tap water to an aquarium . The aquarium was situated in a room with a constant temperature of 16 °C. Immediately after the preparation of the artificial seawater, a total of 90–100 blue mussels were transferred to the aquarium for acclimation for 24 h before 40 mL inoculum of ~2 × 10E. coli overnight broth culture was added and before the blue mussels were harvested 17 h after inoculation. After being removed from the aquarium the mussels were brought to the laboratory on ice. Each was marked with a waterproof marker and kept at 5 °C until steaming. Uninoculated blue mussels with temperature loggers (Signatrol SL53T Temperature logger 0/125 °C) were included in the pot during steaming in order to obtain information on the temperature inside the mussel shells flesh during the cooking period. Water samples were taken after the E. coli from the negative controls. A total of 10 inoculated blue mussels were transferred to a pot with approximately 70–80 not-inoculated blue mussels and steamed for pre-determined periods of time . After steaming, the flesh from the inoculated blue mussels were allowed to cool in room temperature before being distributed in Stomacher bags, one bag per mussel, for quantitative analyses of E. coli as described in E. coli as negative controls and to check the level of E. coli present in the inoculated mussels, respectively.There was no growth of E. coli (2016-01-4162-1-1-1-1 and 2016-01-4220-1-1-1-1) mix . Before sealing, as much air as possible was squeezed out of the bags, and the mussel flesh was not flattened beyond this before heat treatment, retaining the effect of slow heat transfer within the mussel during cooking. The inoculated bags were then stored at 5 °C for at least 30 min prior to heat treatment. The heat treatment was carried out by completely submerging the parts of the bags with blue mussel flesh in a water bath (Nüve BM 30) at fixed temperatures  for pre-determined periods of time (20–270 s). The bags were allowed to cool at room temperature analogous to the whole mussels prior to quantitative analyses for E. coli and ESBL, respectively, as described in For the experiments with the heat treatment of the inoculated flesh from blue mussels, the flesh was obtained from blue mussels purchased from a local supermarket (see above). Aliquots of 10 g were distributed in separate 400 mL Stomacher bags  and inoculated separately with 100 µL of ESBL mix (2016-01-4162-1-3-1-1 and 2016-01-4220-1-3-1-1) or Temperature loggers were included in similar samples with uninoculated blue mussel flesh.E. coli and ESBL E. coli prior to heat treatment to estimate the initial concentration in the mussel flesh. Uninoculated control samples without heat treatment were analyzed for E. coli and ESBL.Inoculated samples were analyzed for E. coli or ESBL-producing E. coli, respectively. In order to obtain a detection limit of 10 cfu/g, one mL of the initial homogenate was distributed equally on the surface of three plates. The TBX and MaC-CO plates were incubated at 37 ± 1 °C and 41.5 ± 1 °C, respectively. Typical colonies on the different agars were counted and a selection of colonies was further confirmed  or Peptone Saline  (heat treatment with inoculated blue mussel flesh) and 100 µL of the appropriate dilutions was plated out with a L-rod on TBX (Oxoid) or MacConkey (Becton Dickinson) supplemented with 1 mg/L cefotaxime (Sigma) (MaC-CO) for quantification of onfirmed .After serial dilutions and plating had been made for quantitative analysis, the rest of the initial homogenate (10 g sample and 90 mL BPW-ISO) from the heat treatment experiment and the aquarium experiment (flesh from one blue mussel diluted 1:10 with BPW-ISO) were incubated at 37 ± 1 °C overnight, followed by plating of a loopful (10 µL) of enrichment on TBX or MaC-CO, depending on which organism analyzed for. All samples were enriched, but plating was only performed if the result from the quantitative analysis was below the detection limit . The plates were inspected for typical colonies and a selection of colonies was pure-cultured on blood agar and further confirmed .E. coli by MALDI Biotyper MS . Presumptive ESBL-producing E. coli were confirmed by using PCR specific for the genes harbored by the strains [A selection of colonies from both the blue mussel inoculation experiment and the heat treatment experiment were confirmed as  strains ,12,13.Data analysis was conducted using the R 3.5 software  with theP of mussels opening so that the estimate proportion EP of mussels opened for a given time point EP, but only after enrichment, E. coli numbers were set to 9 cfu/g.Data were treated to account for “worst case scenarios”, so when When modeling how many bacteria that would remain viable after heat treatment, repeated measures on the same mussel were not feasible. Hence, estimates of contamination level had to be made independently on different individual samples. As a measure Pe of proportional survival of bacteria, this means taking the cfu/g for a given sample at time t minutes of the heat treatment and dividing it by the average cfu/g found in inoculated samples at t = 0, i.e., before any heat above room temperature was applied, rounding any numberThe proportion of the remaining viable cfus were estimated by the GAM regression models with the non-parametric penalized thin spline model with quasi-binomial error distribution to allow for overdispersion and allow the estimation of survival curves to be data-driven and flexible rather than being bound to specific a priori survival functions such as the Weibull  or expon splines  with smoWhen modeling the number of cfu/g directly, not as a proportion remaining of the initial concentration after inoculation, the same model framework was used. Except for the response variableAnd when the conditional distribution of the response is a quasi-Poisson distribution , i.e., wWhen estimating the exposure, we assumed that the observed concentrations of bacteria were representative of an underlying probability density. We then estimated smoothed empirical probability densities on the observed concentrations and simulated expected exposures by drawing 20 hypothetical shells as a typical meal from these distributions, taking the average bacterial concentration (cfu/g) and multiplying by 250, as 250 g is assumed to be a typical portion of blue mussels.For temperature logger data, a set of algorithms was developed to identify and homogenize logger time series, but an element of manual delineating was most efficient as some treatment times had not left peaks identifiable beyond noise and temperature fluctuations between refrigerators, water and air.In traditional preparation, where a culinary value is placed on minimizing heat exposure, looking for the mussel shells opening under steaming is a common indicator for when they can be taken off the heat. The opening rate is well described by a logistic model of opening as a function of the steaming time T, which explains about 70% of the variance in openness status for mussels Methods . The opey-axis values represent the fraction of mussels open . The blue rug lines indicate datapoints along the time axis. Model fit is shown in red, with a 2SE (SE = Standard error) confidence interval for model fit shaded gray.Black squares are averages. CFUP  as an explanatory variable was not significant, suggesting that at these concentrations the survival rate of bacteria was independent of concentration. See Whether or not a mussel is fully open is highly correlated with the proportion Simulating meal exposures from eating 20 mussels  suggests a 10% risk of ingesting a dose over 10% of the original (pre-steaming) concentration of bacteria. If partially opened mussels are included in the meal, the risk increases significantly, as 10% of meals will contain bacteria corresponding to 20% or more of the original concentration in the meal as a whole. See E. coli was not detected in any of the samples. We thus obtained a range of steam times predicted to bring exposure down to regulation levels depending on meal size and contamination level estimated from this. See Modeling the cfu/g directly in an overdispersed Poisson regression model see  using thE. coli strains used in the present study start to be inactivated at temperatures exceeding 55°C. See These steam times map closely to the maximum temperature found by temperature loggers to be attained within steamed mussels and exchanging the max temperature for the time had the same explanatory power. The effect of the steaming time is thus suggested to be largely mediated through the maximum temperature and the time over which it is applied. However, using maximum temperature as an explanatory variable does suggest that the effect of temperature is strongly non-linear and that the E. coli and ESBL producing E. coli, experiments spanning the temperatures relevant for steamed shells were chosen. Mussel flesh was therefore taken out of the shell prior to heating, and the mussel flesh was inoculated with the bacteria.For comparisons of indicator strains of Blue mussel flesh was then inoculated directly and treated at constant temperatures for pre-determined periods. E. coli and indicator E. coli remained in high concentrations even after 270 seconds of heat treatment. Surprisingly, the germination rate seemed possibly even higher after warming. At 55 °C, both ESBL-producing E. coli and ESBL-producing E. coli being detected in some of the samples that were treated for 90 to 240 s, but after 270 s at 65 °C, neither indicator E. coli nor ESBL-producing E. coli could be detected. At 65°C, there was more variation, for both indicator E. coli and ESBL-producing E. coli could be detected in some of the samples up to 100 s treatment. This could be explained by the uneven distribution of the inoculum in the samples. After 110 s at 75 °C, neither indicator E. coli nor ESBL-producing E. coli could be detected. At 75 °C, the mussel flesh samples were generally negative after 40–60 s treatment, but some E. coli, except for one sample from which indicator E. coli was detected after enrichment of the sample (<10 cfu/g).None of the control samples contained indicator lower survival for ESBL E. coli at the very lowest (55 °C) treatment, where bacterial survival rates were nevertheless very high. In general, survival seemed indistinguishable between our genotypes of ESBL E. coli and the indicator strains used here. We also saw that the survival time at the higher temperature treatments was very short, and that models using exposure time, temperature and ESBL status explain approximately 90% of the variance, leaving little noise. See When modeling the effects of heat treatment in water baths at constant temperature see , we saw E. coli and other AMR bacteria behave in raw and lightly cooked conditions. Conditions of stress, such as heat, may trigger several mechanisms in bacterial cells, e.g., adaptation, cellular repair, application of response mechanisms and enhanced virulence [E. coli, Salmonella Typhimurium, Staphylococcus aureus and Cronobacter sakazakii.It is mostly unknown how ESBL-producing irulence . Severalirulence ,21 have E. coli by allowing the blue mussels to acclimatize in artificial seawater in the aquarium prior to adding E. coli. This is, to our knowledge, the first time an experimental study has been carried out to estimate the survival of E. coli as well as ESBLs during the steaming of blue mussels mimicking the cooking procedure in the consumer’s home.For this study we successfully inoculated live blue mussels with E. coli present before steaming is expected to survive if the mussels are only steamed until they are opened and not longer, and that the variation between mussels gives an overall likelihood of about 10% to ingest meals with an exposure corresponding to 10% of the pre-cooking bacterial concentration. It is more if half-opened mussels are included.A notable finding is that the study showed that about 1.5% of the E. coli survived as well as the indicator strains in our other experiments, this means that the traditional preparation method cannot be trusted to inactivate AMR E. coli or other bacteria with similar heat inactivation profiles if they are present in the raw mussels. Considering the mussels ability to concentrate bacteria from the surrounding seawater, and that the time point when >95% are open probably represents an optimistic estimate for how long a consumer would keep heat treating, this suggests that they can be a significant source of human exposure to environmental AMR genotypes present in coastal waters or mussel farms, unless effective monitoring and/or heat treatment practices are in place.As the ESBL E. coli heat tolerance [The inactivation curves found in our water bath experiments seem consistent with previous reports on olerance , where tE. coli followed indistinguishable trajectories under heat treatment, indicating that thermal inactivation curves for E. coli can be used for risk exposure models of resistant isolates, as have been conducted in a recent risk exposure study of ground beef [E. coli O157:H7 for hamburgers [The survival of indicator and ESBL und beef , which umburgers . Neverthmburgers .E. coli. While E. coli is often seen as a heat-sensitive organism, some strains are among the most heat-resistant of foodborne pathogens with D60 values >6 min [55 and D65 in our experiment, we note that our strain seems to be representative of the most commonly tested of E. coli strains [A factor we did not have the opportunity to explore is the differences in heat tolerance between different genotypes of s >6 min ,25,26, w strains , but notAnother factor of concern that needs addressing in this context is the possible transfer of MGEs remaining after thermal inactivation, which cannot be ruled out and needs further studies. Work on post-inactivation MGE transfer and inoculation studies addressing differences in heat tolerance between strains and any possible links between heat resistance and AMR phenotype require further study.The present study has indicated thatShellfish prepared traditionally is a potential pathway for exposure to viable AMR bacteria concentrated from the environment.Inoculation studies mimicking natural bacterial accumulation and realistic preparation have been shown to be feasible and a useful model system.Consuming blue mussels only steamed to opening carries a significant risk of viable bacteria being present in concentrations just one order of magnitude reduced from the raw state.E. coli strains.Steaming time (>3.5–4.0 min) is a better indicator than shell openness to avoid exposure to these ESBL or indicator Further studies including more genotypes and relating them to what is found in the environment are needed.E. coli and an indicator E. coli strain in the studied food matrix.No difference in heat tolerance was found between ESBL